Field of the Invention
The present invention relates to an electrophoresis pattern reading system of a fluorescence type and, more particularly, to a pattern reader for electrophoresis of fluorescence type, in which detachable migration units are mounted to a plurality of electrophoresis units which are electrophoresed simultaneously with each other, and an electrophoresis pattern is read by a common reader unit, thereby efficiently implementing electrophoresis and reading the electrophoresis pattern.
The pattern reader for electrophoresis of the fluorescence type has the advantage that it does not require dangerous and expensive radioisotopes.
Generally speaking, electrophoresis analysis methods using fluorescence method had been used for analysis of various genetic structures including DNA sequencing (determination of a sequence of bases of the gene), mass spectrometry of proteins such as amino acids and analysis of polymer structures. Such an electrophoresis analysis method involves implementing electrophoresis using a sample of fragments labeled with a fluorescent substance and a distribution pattern developed by electrophoresis is analyzed to thereby analyze the samples.
Description will be made of a DNA sequencing device as a representative example of an electrophoresis pattern reader.
In the DNA sequencing using the DNA sequencing device, a sample of a DNA whose structure is determined is first cut into fragments with a restriction enzyme by controlling reactivity against a chemical reaction specific to a site of each a base and labeling them with a fluorescent substance. The fragments are different in length from each other and have each a particular base selected from four kinds of bases labeled at their cut ends, consisting of adenine (A), cytosine (C), guanine (G) and thymine (T). As the fragmented DNA sample can be separated by electrophoresis in accordance with the length of the fragment, each fragment is separated by means of electrophoresis and radiated with laser light to excite the fluorescent substance labeled on each of the fragments. A measurement of the distribution in intensity of the fluorescence emitted from the fluorescent substance permits the reading of a sequence of bases, thereby determining the structure of the DNA.
FIG. 13 is a view showing an example of the distribution of DNA fragments obtained by electrophoresis. As the distance of migration varies with lengths of DNA fragments (the difference of their molecular weights), the fragments having the same molecular weights gather together as time passes, and in an electrophoresis pattern 70, as shown in FIG. 13, bands 66 are formed so as to correspond to the molecular weights of the DNA fragments. As a whole, the electrophoresis pattern is provided such that the bands 66 are formed in lanes 71, 72, 73 and 74. It is to be noted herein that, as there is the difference in molecular weight by one base or more among the bases A, G, C and T of the fragments, the distances of migration for all fragments are different from the other. Hence, it can be theoretically concluded that the bands 66 in the lanes 71 to 74 are not disposed transversely in a row with each other. For DNA sequening, the pattern of the bands 66 is read for each of the bases, A, G, C and T in the respective lanes 71 to 74 in order from the bottom of the pattern, thereby analyzing the DNA sequence.
The above description has been made of the electrophoresis analysis method using the DNA sequencing for analyzing the sequence of the bases of each DNA as an example. It is to be noted, however, that the electrophoresis method can likewise be applied to analysis of other samples. The electrophoresis of the sample subject to analysis allows the sample to be separated into different molecular weights and to form bands corresponding to the separated molecular weights. Hence, the difference in molecular weight between the components of the sample can be determined by reading the distribution of the bands formed. Furthermore, the electrophoresis may be applied to assumption of a molecular weight of a compound or determination of the presence or absence of a given molecule by measuring the distance of migration of the sample and judgment of the presence or absence of the band in a predetermined position.
By pouring a sample labeled with a fluorescent substance into a gel functioning as a base for electrophoresis and electrophoresing the gel, the gel is provided with a distribution of bands after electrophoresis in accordance with the molecular weights of components of the sample so that the distribution of the bands is measured. A measurement of the band distribution is made by radiating the electrophoresed gel with light such as laser light that generates fluorescence upon excitement of the fluorescent substance, and the distribution pattern of the bands is measured by detecting the fluorescence emitted upon reaction with a photoelectrically converting element.
The electrophoresis device of fluorescence detecting type is described, for example, in Japanese Patent Unexamined Publication (kokai) No. 61-62,843/1986.
Description will now be made of the electrophoresis device of such fluorescence type.
FIG. 9 is a perspective view showing an outlook of a conventional electrophoresis device. As shown in FIG. 9, the conventional electrophoresis device comprises an electrophoresis and instrumentation unit 51 for carrying out electrophoresis and instrumenting the distribution of fluorescence, a data processor unit 52 for processing data instrumented, and a cable 53 connecting them to each other. The electrophoresis and instrumentation unit 51 has a door 51a and the door 51a is opened to pour a gel functioning as a base for electrophoresis of DNA fragments and then a given amount of a sample to be analyzed. Then the door 51a is closed and a switch is turned on to start up electrophoresis. As electrophoresis has been started up, an operational state is displayed and monitored on a display panel 51b of the electrophoresis and instrumentation unit 51. The data instrumented is then transferred to the data processor unit 52 and is subjected to desired data processing in accordance with preset programs. The data processor unit 52 comprises predominantly a main body of a computer 54 consisting of a microprocessor, memory and so on, a keyboard 55 from which instructions are given by the operator, a display 56 for display processing results and states, and a printer 57 for recording the processed results.
FIG. 10 is a block diagram showing the construction of the electrophoresis and instrumentation unit. As shown in FIG. 10, the electrophoresis and instrumentation unit 51 (FIG. 9) comprises an electrophoresis subunit 63 and a signal processor subunit 64. The electrophoresis subunit 63 further comprises an electrophoresis section 5 in which electrophoresis is performed, a first electrode 2a and a second electrode 2b for applying voltage to the electrophoresis section 5, a support plate 3 for supporting the electrophoresis section 5 and the first and second electrodes 2a and 2b, a power supply 4 for electrophoresis for applying voltage to the electrophoresis section 5, a light source 11 for generating light to excite a fluorescent substance, an optical fiber 12 for leading the light from the light source 11, a condenser 14 of an optic system for condensing and collecting fluorescence 13 generated by the fluorescent substance, an optical filter 15 for selectively passing the light having a particular wavelength therethrough, and an optical sensor 16 for converting the condensed light into electrical signals. The signal processor subunit 64 further comprises an amplifier 17 for amplifying the electrical signals from the optical sensor 16, an analog-digital converting circuit 18 for converting analog signals of the electrical signals into digital data, a signal processing section 19 for implementing pre-processing of the digital data converted, for example, by addition average processing or the like, an interface 20 for implementing the interface processing for feeding the pre-processed data to an external data processor, and a control circuit 10 for performing an entire control of the electrophoresis and the signal processing. The digital signal OUT is generated from the signal processor subunit 64 and then supplied to the data processor unit 52 (FIG. 9), thereby implementing the data processing such as analysis processing and so on.
Description will now be made of operation of the electrophoresis device which is constructed in the manner as described hereinabove.
Reference is made to FIGS. 9 and 10. After the door 51a of the electrophoresis and instrumentation unit 51, a gel is poured into the electrophoresis section 5 disposed within the unit 51 and thereafter a sample of DNA fragments labeled with a fluorescent substance is poured thereinto. A switch of the display panel 51b is turned on to give an instruction for starting-up electrophoresis, and then voltage is applied from the first and second electrodes 2a and 2b of the power supply 4 to the electrophoresis section 5, thereby starting up electrophoresis. The electrophoresis allows the sample labeled with the fluorescent substance to be migrated in the lanes 71, 72, 73 and 74, thereby gathering the molecules having the same molecular weights together and forming the bands 66, for instance, as shown in FIG. 13. The molecules having smaller molecular weights are allowed to migrate at a rate faster than those having greater molecular weights so that the former is migrated in a distance longer than the latter within the same time unit. The bands 66 are detected in a manner as shown in FIG. 11a by leading light from the light source 11 through the optical fiber 12 and radiating the gel in the electrophoresis section 5 on its optical path, thereby forcing the fluorescent substance labeled on the bands 66 of the gel to emit fluorescence 13.
Referring to the front view as shown in FIG. 11a and to the longitudinally sectional view as shown in FIG. 11b, the electrophoresis section 5 comprises a gel 5a consisting of polyacrylic amide or the like and support plates 5b and 5c made of glass for supporting and interposing the gel 5a from the both sides. For example, a sample of DNA fragments is poured into the gel 5a of the electrophoresis section 5 from its upper portion and electrophoresis is carried out by applying voltage to the first electrode 2a and the second electrode 2b (FIG. 10). Light radiated from the light source, for example, laser light, passes through the light path 61 in the gel 5a from the optical fiber 12 and radiated to the fluorescent substance on the light path 61. This allows the fluorescent substance present on the light path 61 to be excited to emit fluorescence 13. The fluorescence 13 emitted is led to a substage condenser 14 of optics consisting of a combination of lenses and then selected by the optical filter 15 after being condensed, thereby converting it into electrical signals by means of the sensor 16. The electrical signals obtained by the sensor 16 is amplified to a desired level by the amplifier 17 and subjected to analog-digital conversion by the analog-digital circuit 18 followed by a supply to the signal processing section 19. The signal processing section 19 processes the signals by means of addition-average processing or the like in order to improve a signal-noise ratio. The data of the digital signals which has been subjected to signal processing is fed to the data processor subunit 52 through the interface 20.
FIGS. 12a and 12b are views describing an embodiment of fluorescence intensity pattern signals of the DNA fragments to be transferred from the electrophoresis and instrumentation subunit 51. For instance, as shown in FIG. 12a, as the laser light is radiated upon the electrophoresis section 5 in which the electrophoresis is performed, the fluorescent substance of the gel present on the light path 61 is excited to emit fluorescence 13. This fluorescence 13 is detected in predetermined detection positions in each lane in the direction of electrophoresis in the course of lapse of time. This allows the fluorescence 13 to be detected when the bands 66 in each lane pass through the positions on the light path 61, thereby detecting a pattern signal of fluorescence intensity in each lane, as shown in FIG. 12b. Therefore, the pattern signal of the magnitude of fluorescence intensity as shown in FIG. 12b is represented as a pattern signal of fluorescence intensities of the bands 66 in the electrophoresis direction 62. The data processor unit 52 performs data processing for comparing molecular weights and determining the sequence of DNA from data of the pattern of fluorescence intensity. The sequence of the bases or the like determined by data processing is symbolized and then generated, thereby being displayed on a display screen 56 or printed by a printer 57. The data of the result obtained by data processing may be recorded in magnetic recording media as needed. It is to be noted that the time period required for electrophoresis by the pattern reader for electrophoresis having the construction as described hereinabove ranges usually from 5 to 8 hours in the case of electrophoresis of DNA fragments and the time period for reading the distribution of the fluorescent substances in the electrophoresed gel. Hence, it is the current situation in which the analyzer for the electrophoresis method such as the DNA sequencer is occupied for most of its treating time by electrophoresis once the processing for analyzing the substance has been started up. The data processor unit for data processing of the results read from the electrophoresis pattern, which is to be used together with the analyzer for the electrophoresis method of this kind, is constructed as a separate unit so as to allow a general-purpose data processor to be utilized therefor. The electrophoresis and instrumentation unit in which electrophoresis is performed and the electrophoresis pattern is read is an integral combination of an electrophoresis subunit which performs electrophoresis and a signal processor subunit which implements data processing by reading the band pattern as a result of electrophoresis. Hence, once electrophoresis for one sample has started up, the electrophoresis and instrumentation unit is occupied for a long period of time for a series of analyzing processes in which the pattern is read. More specifically, as described hereinabove, for the conventional electrophoresis pattern reading system, the electrophoresis and instrumentation unit has been occupied for the total period of time of about 5-8 hours for electrophoresis and 30 minutes for reading the distribution of the fluorescent substance developed in the gel, so that it is the problem that the expensive system cannot effectively be used.